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Brandon:Protocols/Nextera XT V2 sequences and outline
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===Current sequences and method used by nextera protocol:=== Current nextera sequences i5 adapter 5β- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3' 3'- TCTACACATATTCTCTGTC/5Phos/ -5' i7 adapter 5β- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3' 3'- TCTACACATATTCTCTGTC/5Phos/ -5' After single insertion (Tn5 have only i5 or i7 bound, fragments, 9 base pair gap) i5 adapter i5 adapter NNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN NNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT -5' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNN i7 adapter i7 adapter NNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG NNNNNNNNNNNNN NNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNN After tagementation fragment options: -single insertion not able to make a PCRable fragment -Insertion into bottom strand will just reverse the orientation, i5,i7 will be flipped. i5 adapter i7 adapter 5β- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' i7 adapter i5 adapter 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT -5' i5 adapter i5 adapter 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT -5' i7 adapter i7 adapter 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' After fill in at 72 for 3-5 minutes (gap filling). i5 adapter i7 adapter 5β- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' 3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' i7 adapter i5 adapter 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTGACGCTGCCGACGA -3' 3'- CAGAGCACCCGAGCCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT -5' i5 adapter i5 adapter 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTGACGCTGCCGACGA -3' 3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT -5' i7 adapter i7 adapter 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' 3'- CAGAGCACCCGAGCCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' Now PCRable, adding on dual barcodes. i5 adapter 5β AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC -3' 5β- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' 3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC i7 adapter Amplification with primer 1 and primer 2 (optional) (primer 1) 5'- AATGATACGGCGACCACCGA -3' i5 adapter 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3' 3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' i7 adapter 3'- AGCATACGGCAGAAGACGAAC -5' (primer 2) Nextera read primers for sequencing: i5 index read -----> read1 -----> i7 index read -----> AATGATACGGCGACCACCGAGATCTACAC TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG CTGTCTCTTATACACATCTCCGAGCCCACGAGAC 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3' 3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG <----- read2 num. i5 barcode i5 name i5 smplsheet i5 full sequence 1 CTCTCTAT S502 CTCTCTAT AATGATACGGCGACCACCGAGATCTACACCTCTCTATTCGTCGGCAGCGTC 2 TATCCTCT S503 TATCCTCT AATGATACGGCGACCACCGAGATCTACACTATCCTCTTCGTCGGCAGCGTC 3 GTAAGGAG S505 GTAAGGAG AATGATACGGCGACCACCGAGATCTACACGTAAGGAGTCGTCGGCAGCGTC 4 ACTGCATA S506 ACTGCATA AATGATACGGCGACCACCGAGATCTACACACTGCATATCGTCGGCAGCGTC 5 AAGGAGTA S507 AAGGAGTA AATGATACGGCGACCACCGAGATCTACACAAGGAGTATCGTCGGCAGCGTC 6 CTAAGCCT S508 CTAAGCCT AATGATACGGCGACCACCGAGATCTACACCTAAGCCTTCGTCGGCAGCGTC 7 CGTCTAAT S510 CGTCTAAT AATGATACGGCGACCACCGAGATCTACACCGTCTAATTCGTCGGCAGCGTC 8 TCTCTCCG S511 TCTCTCCG AATGATACGGCGACCACCGAGATCTACACTCTCTCCGTCGTCGGCAGCGTC 9 TCGACTAG S513 TCGACTAG AATGATACGGCGACCACCGAGATCTACACTCGACTAGTCGTCGGCAGCGTC 10 TTCTAGCT S515 TTCTAGCT AATGATACGGCGACCACCGAGATCTACACTTCTAGCTTCGTCGGCAGCGTC 11 CCTAGAGT S516 CCTAGAGT AATGATACGGCGACCACCGAGATCTACACCCTAGAGTTCGTCGGCAGCGTC 12 GCGTAAGA S517 GCGTAAGA AATGATACGGCGACCACCGAGATCTACACGCGTAAGATCGTCGGCAGCGTC 13 CTATTAAG S518 CTATTAAG AATGATACGGCGACCACCGAGATCTACACCTATTAAGTCGTCGGCAGCGTC 14 AAGGCTAT S520 AAGGCTAT AATGATACGGCGACCACCGAGATCTACACAAGGCTATTCGTCGGCAGCGTC 15 GAGCCTTA S521 GAGCCTTA AATGATACGGCGACCACCGAGATCTACACGAGCCTTATCGTCGGCAGCGTC 16 TTATGCGA S522 TTATGCGA AATGATACGGCGACCACCGAGATCTACACTTATGCGATCGTCGGCAGCGTC num. i7 barcode i7 name i7 smplsheet i7 full sequence 1 TCGCCTTA N701 TAAGGCGA CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGG 2 CTAGTACG N702 CGTACTAG CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGG 3 TTCTGCCT N703 AGGCAGAA CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGG 4 GCTCAGGA N704 TCCTGAGC CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGG 5 AGGAGTCC N705 GGACTCCT CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGG 6 CATGCCTA N706 TAGGCATG CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGG 7 GTAGAGAG N707 CTCTCTAC CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGG 8 CAGCCTCG N710 CGAGGCTG CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGG 9 TGCCTCTT N711 AAGAGGCA CAAGCAGAAGACGGCATACGAGATTGCCTCTTGTCTCGTGGGCTCGG 10 TCCTCTAC N712 GTAGAGGA CAAGCAGAAGACGGCATACGAGATTCCTCTACGTCTCGTGGGCTCGG 11 TCATGAGC N714 GCTCATGA CAAGCAGAAGACGGCATACGAGATTCATGAGCGTCTCGTGGGCTCGG 12 CCTGAGAT N715 ATCTCAGG CAAGCAGAAGACGGCATACGAGATCCTGAGATGTCTCGTGGGCTCGG 13 TAGCGAGT N716 ACTCGCTA CAAGCAGAAGACGGCATACGAGATTAGCGAGTGTCTCGTGGGCTCGG 14 GTAGCTCC N718 GGAGCTAC CAAGCAGAAGACGGCATACGAGATGTAGCTCCGTCTCGTGGGCTCGG 15 TACTACGC N719 GCGTAGTA CAAGCAGAAGACGGCATACGAGATTACTACGCGTCTCGTGGGCTCGG 16 AGGCTCCG N720 CGGAGCCT CAAGCAGAAGACGGCATACGAGATAGGCTCCGGTCTCGTGGGCTCGG 17 GCAGCGTA N721 TACGCTGC CAAGCAGAAGACGGCATACGAGATGCAGCGTAGTCTCGTGGGCTCGG 18 CTGCGCAT N722 ATGCGCAG CAAGCAGAAGACGGCATACGAGATCTGCGCATGTCTCGTGGGCTCGG 19 GAGCGCTA N723 TAGCGCTC CAAGCAGAAGACGGCATACGAGATGAGCGCTAGTCTCGTGGGCTCGG 20 CGCTCAGT N724 ACTGAGCG CAAGCAGAAGACGGCATACGAGATCGCTCAGTGTCTCGTGGGCTCGG 21 GTCTTAGG N726 CCTAAGAC CAAGCAGAAGACGGCATACGAGATGTCTTAGGGTCTCGTGGGCTCGG 22 ACTGATCG N727 CGATCAGT CAAGCAGAAGACGGCATACGAGATACTGATCGGTCTCGTGGGCTCGG 23 TAGCTGCA N728 TGCAGCTA CAAGCAGAAGACGGCATACGAGATTAGCTGCAGTCTCGTGGGCTCGG 24 GACGTCGA N729 TCGACGTC CAAGCAGAAGACGGCATACGAGATGACGTCGAGTCTCGTGGGCTCGG
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